[Gly~（14）]-Humanin拮抗淀粉样β-蛋白神经毒作用及其机制的电生理和胞内钙成像研究
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摘要
阿尔茨海默病(Alzheimer’s disease, AD)即老年性痴呆,是一种以进行性学习记忆和认知功能障碍为特征的中枢神经系统退行性疾病。AD的典型病理特征之一是脑内出现高密度的老年斑,其主要成分是由39~43个氨基酸组成的淀粉样β-蛋白(amyloidβ-protein, Aβ)。目前,有关Aβ的神经毒性作用,无论是离体还是在体实验,都已有广泛报道。然而,Aβ发挥神经毒性作用的细胞和分子机制很复杂,迄今为止还不十分清楚,也还缺乏有效的针对Aβ的抗AD药物。
海马长时程增强(long-term potentiation, LTP)是突触传递可塑性的一种表现形式,其损伤与动物认知缺陷高度相关,故海马LTP已被广泛作为研究学习和记忆的电生理细胞模型。有报道表明,AD患者的认知功能下降与中枢突触可塑性如海马LTP受到损伤有关。我们先前的实验也证实,Aβ能够伤害大鼠在体或离体的海马LTP。参与海马LTP诱导的一个重要的受体是N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体,γ-氨基丁酸(gamma-aminobutyric acid,GABA)受体也发挥重要的调制海马LTP的功能。研究表明,在Aβ伤害海马LTP的神经毒性作用中,NMDA和GABAA受体很可能是Aβ的有效靶点。Aβ及其有效活性片段可能通过影响NMDA和GABAA受体的功能影响突触可塑性和海马LTP,从而影响认知功能。同时,大量研究已经证明,Ca2+作为一种第二信使在调节细胞反应、影响神经系统的发育和突触可塑性活动中起关键作用;Aβ诱导的钙内流可能是Aβ引起的众多事件中较早发生的事件,胞内钙稳态的紊乱是Aβ发挥神经毒性作用的重要机制之一。因此,积极寻找拮抗Aβ神经毒性、保护NMDA受体、GABA受体及细胞内Ca2+稳态免受Aβ伤害的措施或药物,成为治疗和预防AD的一个关键问题。
Humanin(HN)及其强效衍生物[Gly14]-Humanin(HNG)的发现为AD和其它记忆损伤疾病的治疗开辟了一条新的道路。HN可有效保护神经元免于多种AD相关损伤引起的细胞死亡,如神经毒性Aβ蛋白。并且,在已报道的抗Aβ神经毒性的神经保护因子中,HN是唯一一种既能抑制Aβ神经毒、又能抑制各种家族性AD基因突变和APP抗体诱发的神经毒性作用的短肽。HNG能够逆转Aβ蛋白诱导的实验动物的记忆损伤,这提示HN是一个很有希望的治疗AD的候选药。然而,直到目前为止,HN的神经保护作用仍缺乏在体和离体的电生理证据,HN是否能够拮抗Aβ造成的LTP及LTP相关离子通道或受体的损伤,以及HN神经保护作用的分子机制仍然不很清楚。
因此,本研究进行了如下三部分工作:(1)利用场电位记录手段,检测了脑室注射HNG对不同Aβ片段抑制在体大鼠海马CA1区LTP的作用,并通过酪氨酸激酶抑制剂Genistein的使用,探讨了HNG发挥神经保护作用的酪氨酸激酶机制;(2)采用全细胞膜片钳技术研究了不同Aβ片段对LTP相关的配体门控通道即NMDA和GABA受体的效应,并进一步观察了HNG是否能对此效应发挥调制或保护作用;(3)使用荧光Ca2+成像技术观察了HNG是否对不同Aβ片段引起的[Ca2+]i升高具有保护作用,并探讨是否有酪氨酸激酶机制参与。在以上工作中,通过使用和比较不同Aβ片段,观察其影响场电位、NMDA受体和GABA受体的效应,也将为我们先前提出的假说即Aβ31-35可能是全长Aβ分子发挥神经毒性作用的更短的活性中心提供进一步的证据。
第一部分:[Gly14]-Humanin保护大鼠在体海马CA1区LTP免受淀粉样β-蛋白的伤害
为研究神经保护肽HNG对不同Aβ片段损伤在体大鼠海马CA1区LTP的保护作用及其可能的酪氨酸激酶机制,本实验将麻醉大鼠固定在立体定位仪上,通过给予海马Schaffer侧支单个电刺激、强直电刺激或双脉冲刺激,在海马CA1区诱发和记录基础的场兴奋性突触后电位(field excitatory postsynaptic potentials, fEPSPs)、强直刺激引起的fEPSPs的长时程增强即LTP以及配对脉冲引起的双脉冲易化(paired pulse facilitation, PPF)。经脑室套管向侧脑室内注射各种Aβ片段、HNG及特异性酪氨酸激酶抑制剂Genistein后,观察了各种药物对基础fEPSPs、LTP以及PPF的影响。
结果显示:(1)侧脑室注射生理盐水(对照组)后,fEPSPs幅度保持稳定;给予高频刺激(high-frequency stimulus, HFS)后,fEPSPs的平均幅度立即上升到215.1±8.6%, HFS后60 min时依然保持在163.3±8.5% (n=8)。这表明,大鼠在体海马LTP被成功诱导。(2)侧脑室注射20 nmol Aβ25-35和Aβ31-35对基础突触传递没有影响,但明显压抑了HFS引起的LTP诱导。在HFS后60 min时,LTP值分别为111.4±8.9% (n=7)和103.3±7.4% (n=6),与对照组相比,明显降低(P<0.01)。(3)侧脑室注射HNG(5 nmol)对基础突触传递和HFS诱导的LTP均无显著影响,HFS后60 min时,LTP值为159.2±6.4% (n=5),与对照组相比,没有显著性统计学差异(P>0.05)。但HNG预处理剂量依赖性拮抗了Aβ25-35对LTP的抑制效应。侧脑室联合注射0.2 nmol, 1 nmol或5 nmol HNG和20 nmol Aβ25-35后,LTP值从单独注射Aβ25-35的111.4±8.9% (n=7)分别增加到114.9±6.4% (n=9,P>0.05), 130.9±6.5% (n=8, P<0.01)和163.7±8.6% (n=7, P<0.01)。(4)与Aβ25-35相似,联合注射5 nmol HNG和20 nmol Aβ31-35也不影响基础突触传递,却逆转了Aβ31-35对LTP的压抑。HFS后60 min时,LTP值为158.6±6.1%(n=5),显著高于单独注射20 nmol Aβ31-35时的103.3±7.4% (n=6, P<0.01)。(5)分别给予或联合给予HNG和Aβ25-35,均未发现双脉冲刺激引起的fEPSPs比值的明显变化。(6) 200 nmol Genistein预处理后,明显减弱了HNG对Aβ25-35诱导的LTP损伤的保护作用,在HFS后60 min时,LTP值从163.7±8.6% (n=7)减小到123.0±7.7% (n=4, P < 0.01),HNG预处理组与正常对照组(164.3±5.3%, n=7)非常接近(P>0.05),Genistein预处理组与Aβ25-35单独给予组(111.4±8.9%, n=7)非常相似。
以上结果显示,Aβ31-35和Aβ25-35具有相似的压抑海马LTP效应,提示Aβ31-35可能是Aβ更短的活性中心;HNG能够剂量依赖性地保护海马LTP免受Aβ引起的损伤,并且酪氨酸激酶通路可能参与了HNG的保护作用,提示应用HNG以及增强酪氨酸激酶活性可能成为治疗AD的有效方法或途径。
第二部分:[Gly14]-Humanin逆转了淀粉样β-蛋白对原代培养大鼠皮层神经元NMDA和GABAA受体电流的效应
兴奋性和抑制性突触活动的适度调节是大脑发挥正常生理功能的前提,兴奋性和抑制性受体/通道在突触传递过程特别是突触可塑性中起着关键作用。本实验在第一部分LTP实验发现的基础上,利用全细胞膜片钳技术,记录了原代培养的大鼠皮层神经元NMDA和GABAA受体介导的全细胞电流。通过重力灌流系统急性给予不同的Aβ片段、HNG及特异性酪氨酸激酶抑制剂Genistein,观察了不同的Aβ片段对神经元兴奋性和抑制性受体电流的效应,研究了神经保护肽HNG是否能对此效应产生调制作用及其可能的酪氨酸激酶机制。
实验结果显示:(1)单独应用高浓度(10μM)的Aβ25-35、Aβ31-35或Aβ35-31,均不能诱导出任何明显的跨膜电流。(2)急性给予0.1μM, 1μM和10μM Aβ25-35和Aβ31-35,可逆性地抑制了NMDA诱导的全细胞内向电流(INMDA),并表现出浓度依赖性,使Aβ25-35预处理组INMDA分别减小到86.0±6.7% (n=14, P<0.01), 74.9±8.2% (n=15, P<0.01)和61.0±10.6% (n=13, P<0.01);使Aβ31-35预处理组INMDA分别减小到82.6±8.0% (n=11, P<0.01), 72.1±10.8% (n=14, P<0.01)和60.6±7.8% (n=14, P<0.01),与给药前的(100%)相比均有统计学差异。但是,相同浓度的Aβ25-35和Aβ31-35抑制INMDA的效应没有显著性差异(P>0.05)。(3) HNG剂量依赖地逆转了10μM Aβ25-35和Aβ31-35对INMDA的抑制效应,INMDA从Aβ25-35单独给予时的61.0±10.6% (n=13)改变为共同给予0.1 nM, 1 nM和10 nM HNG时的58.1±5.5% (n=15, P.>0.05),80.7±8.8% (n=12, P<0.01)和96.5±6.0% (n=12, P<0.01);从Aβ31-35单独给予时的60.6±7.8% (n=14)改变为共同给予0.1 nM, 1 nM和10 nM HNG时的56.5±6.2% (n=15, P>0.05), 74.3±4.5% (n=14, P<0.01)和93.9±11.0% (n=13, P<0.01)。(4) 0.1μM, 1μM和10μMAβ25-35和Aβ31-35预处理,可逆性地抑制了GABA诱导的全细胞电流(IGABA),并表现出浓度依赖性。Aβ25-35预处理后IGABA分别减小到96.2±7.3% (n=12, P>0.05), 67.7±6.8% (n=14, P<0.01)和55.5±12.2% (n=13, P<0.01);Aβ31-35预处理后IGABA分别减小到91.0±8.0% (n=11, P<0.05), 71.3±9.1% (n=21, P<0.01)和58.2±9.7% (n=19, P<0.01),与给药前的对照(100%)相比都有统计学差异。同时,Aβ25-35和Aβ31-35抑制IGABA的效应,在相同浓度组之间也未发现显著性差异。(5) HNG剂量依赖性逆转了1μM Aβ25-35和Aβ31-35诱导的IGABA抑制效应。IGABA从Aβ25-35单独给予时的67.7±6.8%增加到共同给予0.01 nM,0.1 nM,1 nM和10 nM HNG时的67.8±9.6% (n=14, P>0.05), 80.8±10.8% (n=12, P<0.01), 90.2±10.9 (n=12, P<0.01)和99.6±9.4% (n=16, P<0.01);从Aβ31-35单独给予时的71.3±9.1%增加到共同给予0.01 nM,0.1 nM,1 nM和10 nM HNG时的66.9±8.8% (n=14, P>0.05), 83.8±8.4% (n=11, P<0.01), 92.2±8.8% (n=12, P<0.01)和102.5±9.7% (n=15, P<0.01)。(6) Aβ25-35和Aβ31-35对较高浓度(100μM,1000μM)GABA引起的全细胞电流没有显著影响。10μM,20μM或40μM Aβ25-35预处理后,100μM GABA引起的IGABA分别维持在99.9±5.9% (n=12, P>0.05),96.1±7.2% (n=11, P>0.05)和98.3±5.6% (n=12, P>0.05);类似地,10μM,20μM或40μM Aβ31-35预处理后,IGABA分别为97.8±5.3% (n=18, P>0.05), 99.3±9.5% (n=12, P>0.05)和95.1±9.6% (n=13, P>0.05)。与给药前(100%)相比均无显著性统计学差异。这表明,高浓度(100μM以上)GABA诱导的IGABA不易受到Aβ的抑制作用。(7)特异性酪氨酸激酶抑制剂Genistein (100μM)预处理后,几乎完全去除了HNG (10 nM)对Aβ25-35和Aβ31-35抑制INMDA和IGABA的保护作用。INMDA分别减小为63.0±8.0% (n=14)和58.9±4.8% (n=14);IGABA分别减小为70.5±11.4% (n=10)和70.5±11.4% (n=10)。与共同给予HNG和Aβ25-35/Aβ31-35相比有统计学差异,但与单独给予Aβ25-35或Aβ31-35相比无显著性统计学差异。(8)给予Aβ31-35的反序列即Aβ35-31预处理后,NMDA或GABA诱发的电流未出现明显改变。以上结果表明:急性给予Aβ25-35和Aβ31-35均可对原代培养的大鼠皮层神经元NMDA和GABA受体产生调制作用。这些结果可能有助于解释Aβ诱导的海马LTP的损害,其中突触内NMDA受体的下调可能会直接影响LTP的诱导;GABA受体的下调有可能破坏神经系统兴奋和抑制的平衡,增加细胞的兴奋毒作用。该结果为Aβ损伤谷氨酸能和GABA能系统以及HNG对抗Aβ神经毒性作用的保护效应提供了进一步的电生理证据。
第三部分:[Gly14]-Humanin对淀粉样β-蛋白引起的原代培养大鼠皮层神经元细胞内钙水平的影响
本实验使用激光扫描共聚焦显微镜成像系统进行细胞内Ca2+荧光成像实验,观察了各种Aβ片段对原代培养的大鼠皮层神经元细胞内Ca2+浓度(intracellular calciumconcentration, [Ca2+]i)的影响,并研究了HNG对Aβ诱导的钙稳态紊乱的保护作用及可能的酪氨酸激酶机制。
实验结果显示:(1) Aβ25-35或Aβ31-35均显著增加了原代培养的皮层神经元细胞内钙水平。细胞外给予25μM Aβ25-35或Aβ31-35处理18分钟后,神经元的相对荧光密度由给药前的对照值(100%)分别明显上升到174.3±10.7% (n=25, P<0.01)和172.7±6.6% (n=40, P<0.01);Aβ25-35和Aβ31-35升高[Ca2+]i的效应,在相同浓度时没有发现显著性差异;给予Aβ31-35的反序列Aβ35-31后,皮层神经元[Ca2+]i没有明显变化。(2) HNG阻断了Aβ25-35和Aβ31-35诱导的[Ca2+]i升高,并表现出一定程度的剂量依赖性。我们发现,用不同浓度的HNG预处理培养的神经元25～30 min后,随着HNG浓度的升高,Aβ25-35或Aβ31-35诱导的[Ca2+]i升高效应逐渐减小。在Aβ25-35组,0.1 nM, 1 nM, 10 nM和100 nM HNG预处理后,Aβ25-35 (25μM)引起的相对荧光密度由单独给予Aβ25-35时的174.3±10.7% (n=25)分别下降为173.6±9.0% (n=21, P>0.05)、144.6±8.2% (n=39, P<0.01)、120.0±6.3% (n=38, P<0.01)和105.4±6.0% (n=33, P<0.01);在Aβ31-35组,以上四种浓度的HNG预处理后,Aβ31-35 (25μM)引起的相对荧光密度则由单独给予Aβ31-35时的172.7±6.6% (n=40)分别下降为164.4±8.8% (n=16, P<0.01)、137.1±6.7% (n=19, P<0.01)、114.4±9.9% (n=23, P<0.01)和103.1±6.2% (n=29, P<0.01)。同单独使用Aβ25-35或Aβ31-35相比,联合使用HNG和Aβ25-35或Aβ31-35引起的[Ca2+]i下降具有明显差别。(3)特异性酪氨酸激酶抑制剂Genistein (100μM)预处理后,基本上去除了HNG (10 nM)对Aβ25-35或Aβ31-35 (25μM)引起的[Ca2+]i升高效应的抑制作用。与联合给予HNG和Aβ25-35/Aβ31-35相比,共同给予Genistein、HNG和Aβ25-35/Aβ31-35后,相对荧光密度值分别从120.0±6.3% (n=38)和114.4±9.9% (n=23)增加到178.3±9.9% (n=29, P<0.01)和168.7±8.2% (n=27, P<0.01),并且与单独给予Aβ25-35或Aβ31-35的相对荧光密度值相似,意味着HNG的保护作用被酪氨酸激酶抑制剂所阻断。
钙成像实验结果表明:(1) Aβ25-35和Aβ31-35均可使原代培养的大鼠皮层神经元[Ca2+]i升高,提示Aβ的神经毒性与Aβ引起的细胞内钙超载有关;(2) HNG剂量依赖地抑制了Aβ25-35和Aβ31-35诱导的[Ca2+]i升高,提示HNG对原代培养的大鼠皮层神经元具有保护作用,其机制至少部分是通过减弱Aβ引起的细胞内钙超载实现的;(3)特异性酪氨酸激酶抑制剂Genistein去除了HNG对Aβ25-35或Aβ31-35引起的[Ca2+]i升高的抑制作用,提示某些酪氨酸激酶参与了HNG对Aβ诱导的[Ca2+]i升高的保护作用;(4) Aβ25-35和Aβ31-35引起[Ca2+]i升高的效应在许多实验组中都非常相似,包括Aβ片段单独使用和Aβ与HNG、Genistein联合使用,这支持我们之前提出的假设:Aβ31-35是全长Aβ分子中一个更短的片段,可能是Aβ分子发挥神经毒性作用的活性中心。
总之,本研究利用电生理技术即细胞外场电位记录和全细胞膜片钳记录手段,结合激光扫描共聚焦显微镜Ca2+荧光成像技术,通过记录大鼠在体海马LTP、引导原代培养的皮层神经元NMDA受体和GABA受体电流以及测定神经元细胞内钙离子水平的变化,探讨了神经保护肽HNG对Aβ神经毒性作用的调制以及可能的酪氨酸激酶机制。研究结果首次表明,HNG能够保护神经毒性Aβ诱导的在体海马LTP损伤、逆转Aβ对LTP相关受体(NMDA和GABA受体)电流的损害、并且抑制了Aβ引起的[Ca2+]i升高。我们进一步的结果还提示,酪氨酸激酶通路可能参与了HNG的神经保护作用。因此,本研究为HN的神经保护作用及其机制提供了强有力的电生理和细胞内钙成像证据,为HNG有可能成为AD治疗措施之一提供了研究基础。
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elderly leading to progressive loss of memory and cognitive deficits. One of the predominant neuropathological features of AD is the presence of high density of senile plaques in the brain. The main constituent of senile plaques is amyloidβ-protein (Aβ), which consisting of 39-43 amino acids and coming from the proteolysis of amyloid precursor protein (APP). The neurotoxicity of Aβhas been widely reported in vivo and in vitro, including the impairment of synaptic plasticity, such us long-term potentiation (LTP). The mechanisms underlying Aβ-neurotoxicity are complex and unclear so far but may involve the vulnerability of glutamatergic and GABAergic system. Considerable evidences suggest that Aβand its peptide fragments influence cellular homeostasis and neuronal signaling through modulation of ion channel function.
Hippocampus in the central nervous system has been widely considered to be a crucial center for learning and memory. Hippocampal LTP is an activity-dependent increase in the synaptic response and has been accepted as a popular electrophysiological model for the cellular basis of learning and memory, owing to the close relationship between the hippocampal LTP impairment and the animal cognitive deficient. Further, N-methyl-D-aspartate (NMDA) receptor plays a pivotal role in the LTP induction of CA1 pyramidal neurons because of its high permeability to Ca2+ ions; changes in the efficacy of synaptic inhibition mediated by GABAA receptors are believed to play central roles in certain forms synaptic plasticity, and some reports indicate the potential role of Aβin the deficits of the GABAergic system in the development and pathology progresses of AD. Hence the modulation of LTP-related receptors such as NMDA and GABA receptors is a potential target for the neurotoxicity of Aβon LTP. At the same time, calcium ion is one of the most important intracellular second messengers in the brain, being essential for a variety of neuronal functions such as neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways. It is well known that Aβinduced Ca2+ influx is proposed as the initial event of Aβ-induced multiple events. Although the mechanisms by which Aβincreased [Ca2+]i are still not well known, the disruption of Ca2+ homeostasis is the primary event in Aβneurotoxicity. Thus, it will be very important to search for effective medicines or measures that can protect NMDA receptors, GABA receptors, and intracellular Ca2+ homeostasis against Aβ-induced neurotoxicity in the prevention and treatment of AD.
An important clue in the development of AD therapy is the finding of humanin (HN), a novel short polypeptide, and its potent derivative [Gly14]-humanin (HNG). HN can prevent neuronal cell death caused by various AD-relevant insults such as neurotoxic Aβprotein, and is the only factor that is effective in suppressing various types of AD-related neuronal death so far compared with many neurotrophic factors. The fact that HNG reverses the impairment of memory induced by Aβpeptide suggests that HNG is a promising candidate in the treatment of AD. However, it is still short of in vivo and in vitro electrophysiological evidence for the protection of HN on synaptic plasticity such as LTP and LTP-related postsynaptic receptors/channels, and the molecular mechanisms that underling the neuroprotective function of HN also remain largely unknown.
Therefore, the purposes of the present study were as follows: (1) Examining the effects of intracerebroventricular (i.c.v.) injection of HNG on the different Aβfragments-induced suppression of LTP in the rat hippocampal CA1 region in vivo with field potential recording technique; (2) Investigating whether HNG affects the effects of different Aβfragments on LTP induction-related postsynaptic ligand-gated excitatory receptor channels (NMDAR) and inhibitory receptor channels (GABAR) by using whole-cell patch clamp technique in cultured primary rat cortical neurons; (3) Observing the effects of different Aβfragments on [Ca2+]i, and investigating the possible neuroprotective effects of HNG against Aβ-induced disruption of Ca2+ homeostasis in cultured primary rat cortical neurons by using calcium imaging via laser-scanning confocal imaging system; (4) Clarifying whether tyrosine kinases are involved in the neuroprotective function of HNG by using a specific tyrosine kinase inhibitor, Genistein; and (5) Identifying whether Aβ31-35 is a shorter active center than Aβ25-35 in full length of Aβmolecule by comparing the effects of Aβfragments in the experiments of LTP, whole-cell currents and [Ca2+]i imaging.
PartⅠ:[Gly14]-Humanin Rescues Long-Term Potentiation from AmyloidβProtein-Induced Impairment in the Rat Hippocampal CA1 Region In Vivo
The present study observed the effects of intracerebroventricular (i.c.v.) injection of HNG on the different Aβfragments-induced suppression of hippocampal LTP in area CA1 of urethane anesthetized rats, and explored the probable tyrosine kinase mechanism by which HNG protect Aβ-induced LTP impairment. The results showed that: (1) The amplitudes of the field excitatory postsynaptic potentials (fEPSPs) in control group (i.c.v. injection of saline) increased to 215.1±8.6% immediately after applying high-frequency stimulus (HFS), as compared to that measured before HFS and set arbitrarily as 100%, and remains at 163.3±8.5% 60 min after HFS (n=8). (2) i.c.v. injection of 20 nmol Aβ25-35 and Aβ31-35 had no effect on baseline synaptic transmission, but both of them similarly and significantly depressed the induction of LTP, with fEPSPs of 111.4±8.9% (n=7) and 103.3±7.4% (n=6) at 60 min after HFS, respectively. (3) Application of 5 nmol HNG alone caused no significant effect on baseline synaptic transmission and HFS-induced LTP compared with control, and the LTP at 60 min post HFS was 159.2±6.4%(n=5), which is close to the value of 163.3±8.5% (n=8) found in control group (P>0.05). However, co-injection of HNG and Aβ25-35 reversed the depression of LTP by Aβ25-35 alone in a dose-dependent manner. After i.c.v. co-injection of 0.2 nmol, 1 nmol and 5 nmol HNG with 20 nmol Aβ25-35, the LTP at 60 min post HFS increased from 111.4±8.9% (n=7) in Aβ25-35 alone group to 114.9±6.4% (n=9, P>0.05), 130.9±6.5% (n=8, P<0.01) and 163.7±8.6% (n=7, P<0.01) in three HNG groups, respectively. (4) Similar to Aβ25-35, co-injection of 5 nmol HNG with 20 nmol Aβ31-35 did not affect baseline synaptic transmission, but effectively reversed Aβ31-35-induced LTP suppression, and the LTP value was 158.6±6.1% (n=5) 60 min after HFS, significantly larger than the effect when using 20 nmol Aβ31-35 alone (103.3±7.4%, n=6, P<0.01). (5) Separate application or co-application of HNG and Aβ25-35 showed no significant effect on paired pulse-evoked facilitation. (6) Pretreatment with 200 nmol Genistein obviously attenuated the protection of HNG against Aβ25-35-induced LTP impairment, and the LTP value decreased from 163.7±8.6% (n=7) to 123.0±7.7% (n=4, P<0.01) 60 min after HFS. The former was very close to the value in normal control group (164.3±5.3%, n=7, P>0.05), while the later has been close to the value in Aβ25-35 alone group (111.4±8.9%, n=7, P>0.05).
These results demonstrate that: (1) Aβ31-35 may be a shorter active fragment of Aβbecause of the similar effects of Aβ31-35 and Aβ25-35; (2) HNG could dose-dependently protect against the neurotoxic Aβ-induced hippocampal LTP impairment; and (3) the tyrosine kinase pathway was involved in the neuroprotective action of HNG. All of these results suggest that HNG might be one of the promising candidates for the treatment of AD in the future.
Part II:[Gly14]-Humanin Abolishes the Effects of AmyloidβProtein on NMDA and GABAA Receptor-Mediated Currents in Cultured Primary Rat Cortical Neurons
The proper modulation of excitatory and inhibitory synaptic transmission is critical for brain function including synaptic plasticity such as LTP. The present study extended our investigation of in vivo LTP by observing whether HNG can modulate the effects of different Aβ fragments on LTP induction-related postsynaptic ligand-gated excitatory receptor channels (NMDAR) and inhibitory receptor channels (GABAR) by using whole-cell patch clamp in cultured primary rat cortical neurons. The results showed that: (1) Acute application of Aβ25-35, Aβ31-35 or Aβ35-31 alone did not induce any membrane current, even at a higher concentration (10μM). (2) The NMDA-induced whole cell currents (INMDA) were reversibly inhibited by acute application of 0.1μM, 1μM and 10μM Aβ25-35 and Aβ31-35 in a dose-dependent manner. INMDA decreased to 86.0±6.7% (n=14, P<0.01), 74.9±8.2% (n=15, P<0.01) and 61.0±10.6% (n=13, P<0.01) in Aβ25-35 groups, 82.6±8.0% (n=11, P<0.01), 72.1±10.8% (n=14, P<0.01) and 60.6±7.8% (n=14, P<0.01) in Aβ31-35 groups compared with 100% in control, respectively. (3) HNG dose-dependently reversed the inhibitory effect of Aβ25-35 and Aβ31-35 (10μM) on INMDA. INMDA changed from 61.0±10.6% (n=13) in Aβ25-35 alone group to 58.1±5.5% (n=15, P>0.05), 80.7±8.8% (n=12, P<0.01) and 96.5±6.0% (n=12, P<0.01) at 0.1 nM, 1 nM and 10 nM HNG, respectively; and from 60.6±7.8% (n=14) in Aβ31-35 alone group to 56.5±6.2% (n=15, P>0.05), 74.3±4.5% (n=14, P<0.01) and 93.9±11.0% (n=13, P<0.01), respectively. (4) Meanwhile, pretreatment with 0.1μM, 1μM and 10μM Aβ25-35 and Aβ31-35 reversibly suppressed the GABA-induced whole-cell currents (IGABA) in a concentration-dependent manner. IGABA decreased to 96.2±7.3% (n=12, P>0.05), 67.7±6.8% (n=14, P<0.01) and 55.5±12.2% (n=13, P<0.01) in Aβ25-35 groups, 91.0±8.0% (n=11, P<0.05), 71.3±9.1% (n=21, P<0.01) and 58.2±9.7% (n=19, P<0.01) in Aβ31-35 groups compared with 100% in control, respectively. (5) HNG concentration-dependently reversed Aβ-induced suppression. IGABA changed from 67.7±6.8% in Aβ25-35 alone group to 67.8±9.6% (n=14, P>0.05), 80.8±10.8% (n=12, P<0.01), 90.2±10.9 (n=12, P<0.01) and 99.6±9.4% (n=16, P<0.01) at 0.01 nM, 0.1 nM,? 1 nM and 10 nM HNG, respectively. IGABA changed from 71.3±9.1% in Aβ31-35 alone group to 66.9±8.8% (n=14, P>0.05), 83.8±8.4% (n=11, P<0.01), 92.2±8.8% (n=12, P<0.01) and 102.5±9.7% (n=15, P<0.01) at 0.01 nM, 0.1 nM,? 1 nM and 10 nM HNG pretreatment, respectively. (6) Aβ25-35 and Aβ31-35 have no significant effect on the higher concentration (100μM and 1000μM) of GABA-induced whole cell currents. 100μM GABA induced IGABA were still 99.9±5.9% (n=12, P>0.05), 96.1±7.2% (n=11, P>0.05) and 98.3±5.6% (n=12, P>0.05) after application of 10μM, 20μM or 40μM Aβ25-35. Similarly, after pretreatment with 10μM, 20μM or 40μM Aβ31-35, 100μM GABA induced IGABA were still 97.8±5.3% (n=18, P>0.05), 99.3±9.5% (n=12, P>0.05) and 95.1±9.6% (n=13, P>0.05), respecitively. (7) Pretreatment with Genistein (100μM), a tyrosine kinase inhibitor, nearly completely abolished the protective action of HNG (10 nM) on Aβ25-35 and Aβ31-35 induced inhibition of INMDA and IGABA, the values being 63.0±8.0% (n=14, P<0.01) and 58.9±4.8% (n=14) for INMDA, 70.5±11.4% (n=10) and 70.5±11.4% (n=10) for IGABA. (8) Aβ35-31, the reversed sequence of Aβ31-35, showed no effect on the currents induced by NMDA or GABA.
In conclusion, the present study showed an acute modulation of Aβ25-35 and Aβ31-35 on NMDA receptors and GABAA receptors in cultured primary rat cortical neurons. These results may partly explain the reason of Aβ-induced suppression of hippocampal LTP, in which synaptic NMDA receptor might play important roles. In addition, our results provide an electrophysiological evidence for the Aβ-neurotoxicity on the glutamatergic/GABAergic system and the neuroprotective function of HNG on Aβ-neurotoxicity. The neuroprotection of HNG strongly suggests the potential clinical applications of HNG in novel AD therapies.
PartⅢ:Effects of [Gly14]-Humanin on AmyloidβProtein-Induced Calcium Influx in Cultured Primary Rat Cortical Neurons
The present study, by utilizing calcium imaging via laser-scanning confocal imaging system, observed the effects of different Aβfragments on [Ca2+]i, and investigated the neuroprotective effects of HNG against Aβ-induced disruption of Ca2+ homeostasis and its possible tyrosine kinase mechanism in cultured primary rat cortical neurons. The results showed that: (1) Both Aβ25-35 and Aβ31-35 significantly increased the intracellular calcium levels of the cultured primary rat cortical neurons. After application of 25μM Aβ25-35 or Aβ31-35 to cultured primary rat cortical neurons, the relative fluorescent intensity of neurons obviously increased from 100% in control to 174.3±10.7% (n=25, P<0.01) and 172.7±6.6% (n=40, P<0.01), respectively. However, 25μM Aβ35-31, the reverse peptide of Aβ31-35, had no effect on [Ca2+]i elevation. In addition, the effect of Aβ25-35 and Aβ31-35 on [Ca2+]i elevation, at the same concentration, does not show any statistical difference. (2) Neuroprotective polypeptide HNG blocked the [Ca2+]i elevation induced by Aβ25-35 or Aβ31-35 in a concentration-dependent manner. With the increasing of HNG concentration, Aβ25-35- or Aβ31-35-induced elevation in [Ca2+]i decreased. After pretreatment with 0.1 nM, 1 nM, 10 nM and 100 nM HNG, Aβ25-35-evoked relative fluorescent intensity decreased from 174.3±10.7% (n=25) to 173.6±9.0% (n=21, P>0.05), 144.6±8.2% (n=39, P<0.01), 120.0±6.3% (n=38, P<0.01), and 105.4±6.0% (n=33, P<0.01), respectively. Aβ31-35-evoked relative fluorescent intensity decreased from 172.7±6.6% (n=40) in Aβ31-35 alone to 164.4±8.8% (n=16, P<0.01), 137.1±6.7% (n=19, P<0.01), 114.4±9.9% (n=23, P<0.01) and 103.1±6.2% (n=29, P<0.01), respectively. (3) Pretreatment with Genistein (100μM), a specific tyrosine kinase inhibitor, essentially abolished the protective effect of HNG (10 nM) on Aβ25-35 or Aβ31-35 induced [Ca2+]i elevation. Compared with co-application of HNG and Aβ25-35 or Aβ31-35, the relative fluorescent intensity in co-application of Genistein, HNG and Aβ25-35 or Aβ31-35 increased to 178.3±9.9% (n=29, P<0.01) and 168.7±8.2% (n=27, P<0.01) from 120.0±6.3% (n=38) and 114.4±9.9% (n=23), respectively, similar to the values in Aβ25-35 or Aβ31-35 alone group.
These results demonstrate that: (1) Both Aβ25-35 and Aβ31-35 can similarly elevate [Ca2+]i in cultured primary rat cortical neurons, suggesting that Aβ-neurotoxicity is related with Aβ-induced intracellular calcium overloading. (2) HNG can inhibit Aβ25-35 and Aβ31-35-induced elevation of [Ca2+]i in a concentration-dependent manner, suggesting that the protective action of HNG on the cultured primary rat cortical neurons was, at least in part, mediated by attenuating the intracellular calcium overloading induced by Aβ. (3) Genistein, a specific tyrosine kinase inhibitor, abolished the suppression of HNG on Aβ25-35 or Aβ31-35 induced [Ca2+]i elevation, indicating that certain tyrosine kinase(s) is (are) involved in the protective effects of HNG on Aβinduced [Ca2+]i elevation. (4) The similar [Ca2+]i elevation effects of Aβ31-35 and Aβ25-35 in various groups, including Aβfragments alone and Aβplus HNG and Genistein, supports the hypothesis we proposed previously that Aβ31-35 may be a more shorter fragment of the full length of Aβmolecule.
In conclusion, the present study, by using field potential recording and whole-cell patch clamp technique, observed the modulation of HNG on the effects of Aβfragments on hippocampal LTP in vivo, INMDA and IGABA in cultured primary rat cortical neurons. The possible mechanisms underlying the neuroprotection of HNG were also explored by using Genistein and Ca2+ image technique. The results demonstrated for the first time that HNG could protect against the neurotoxic Aβ-induced hippocampal LTP deficit, reverse the impairment of LTP-related NMDA and GABA receptor currents by Aβand inhibit Aβ-induced [Ca2+]i elevation. Moreover, our results indicated that the neuroprotective actions of HNG may be involved in the tyrosine kinase pathway. Taken all together, all of our experiments in vivo and in vitro provide strong electrophysiological evidences for the neuroprotection of HN and suggest that HNG might be one of the promising candidates for the treatment of AD in the future.
海马长时程增强(long-term potentiation, LTP)是突触传递可塑性的一种表现形式,其损伤与动物认知缺陷高度相关,故海马LTP已被广泛作为研究学习和记忆的电生理细胞模型。有报道表明,AD患者的认知功能下降与中枢突触可塑性如海马LTP受到损伤有关。我们先前的实验也证实,Aβ能够伤害大鼠在体或离体的海马LTP。参与海马LTP诱导的一个重要的受体是N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体,γ-氨基丁酸(gamma-aminobutyric acid,GABA)受体也发挥重要的调制海马LTP的功能。研究表明,在Aβ伤害海马LTP的神经毒性作用中,NMDA和GABAA受体很可能是Aβ的有效靶点。Aβ及其有效活性片段可能通过影响NMDA和GABAA受体的功能影响突触可塑性和海马LTP,从而影响认知功能。同时,大量研究已经证明,Ca2+作为一种第二信使在调节细胞反应、影响神经系统的发育和突触可塑性活动中起关键作用;Aβ诱导的钙内流可能是Aβ引起的众多事件中较早发生的事件,胞内钙稳态的紊乱是Aβ发挥神经毒性作用的重要机制之一。因此,积极寻找拮抗Aβ神经毒性、保护NMDA受体、GABA受体及细胞内Ca2+稳态免受Aβ伤害的措施或药物,成为治疗和预防AD的一个关键问题。
Humanin(HN)及其强效衍生物[Gly14]-Humanin(HNG)的发现为AD和其它记忆损伤疾病的治疗开辟了一条新的道路。HN可有效保护神经元免于多种AD相关损伤引起的细胞死亡,如神经毒性Aβ蛋白。并且,在已报道的抗Aβ神经毒性的神经保护因子中,HN是唯一一种既能抑制Aβ神经毒、又能抑制各种家族性AD基因突变和APP抗体诱发的神经毒性作用的短肽。HNG能够逆转Aβ蛋白诱导的实验动物的记忆损伤,这提示HN是一个很有希望的治疗AD的候选药。然而,直到目前为止,HN的神经保护作用仍缺乏在体和离体的电生理证据,HN是否能够拮抗Aβ造成的LTP及LTP相关离子通道或受体的损伤,以及HN神经保护作用的分子机制仍然不很清楚。
因此,本研究进行了如下三部分工作:(1)利用场电位记录手段,检测了脑室注射HNG对不同Aβ片段抑制在体大鼠海马CA1区LTP的作用,并通过酪氨酸激酶抑制剂Genistein的使用,探讨了HNG发挥神经保护作用的酪氨酸激酶机制;(2)采用全细胞膜片钳技术研究了不同Aβ片段对LTP相关的配体门控通道即NMDA和GABA受体的效应,并进一步观察了HNG是否能对此效应发挥调制或保护作用;(3)使用荧光Ca2+成像技术观察了HNG是否对不同Aβ片段引起的[Ca2+]i升高具有保护作用,并探讨是否有酪氨酸激酶机制参与。在以上工作中,通过使用和比较不同Aβ片段,观察其影响场电位、NMDA受体和GABA受体的效应,也将为我们先前提出的假说即Aβ31-35可能是全长Aβ分子发挥神经毒性作用的更短的活性中心提供进一步的证据。
第一部分:[Gly14]-Humanin保护大鼠在体海马CA1区LTP免受淀粉样β-蛋白的伤害
为研究神经保护肽HNG对不同Aβ片段损伤在体大鼠海马CA1区LTP的保护作用及其可能的酪氨酸激酶机制,本实验将麻醉大鼠固定在立体定位仪上,通过给予海马Schaffer侧支单个电刺激、强直电刺激或双脉冲刺激,在海马CA1区诱发和记录基础的场兴奋性突触后电位(field excitatory postsynaptic potentials, fEPSPs)、强直刺激引起的fEPSPs的长时程增强即LTP以及配对脉冲引起的双脉冲易化(paired pulse facilitation, PPF)。经脑室套管向侧脑室内注射各种Aβ片段、HNG及特异性酪氨酸激酶抑制剂Genistein后,观察了各种药物对基础fEPSPs、LTP以及PPF的影响。
结果显示:(1)侧脑室注射生理盐水(对照组)后,fEPSPs幅度保持稳定;给予高频刺激(high-frequency stimulus, HFS)后,fEPSPs的平均幅度立即上升到215.1±8.6%, HFS后60 min时依然保持在163.3±8.5% (n=8)。这表明,大鼠在体海马LTP被成功诱导。(2)侧脑室注射20 nmol Aβ25-35和Aβ31-35对基础突触传递没有影响,但明显压抑了HFS引起的LTP诱导。在HFS后60 min时,LTP值分别为111.4±8.9% (n=7)和103.3±7.4% (n=6),与对照组相比,明显降低(P<0.01)。(3)侧脑室注射HNG(5 nmol)对基础突触传递和HFS诱导的LTP均无显著影响,HFS后60 min时,LTP值为159.2±6.4% (n=5),与对照组相比,没有显著性统计学差异(P>0.05)。但HNG预处理剂量依赖性拮抗了Aβ25-35对LTP的抑制效应。侧脑室联合注射0.2 nmol, 1 nmol或5 nmol HNG和20 nmol Aβ25-35后,LTP值从单独注射Aβ25-35的111.4±8.9% (n=7)分别增加到114.9±6.4% (n=9,P>0.05), 130.9±6.5% (n=8, P<0.01)和163.7±8.6% (n=7, P<0.01)。(4)与Aβ25-35相似,联合注射5 nmol HNG和20 nmol Aβ31-35也不影响基础突触传递,却逆转了Aβ31-35对LTP的压抑。HFS后60 min时,LTP值为158.6±6.1%(n=5),显著高于单独注射20 nmol Aβ31-35时的103.3±7.4% (n=6, P<0.01)。(5)分别给予或联合给予HNG和Aβ25-35,均未发现双脉冲刺激引起的fEPSPs比值的明显变化。(6) 200 nmol Genistein预处理后,明显减弱了HNG对Aβ25-35诱导的LTP损伤的保护作用,在HFS后60 min时,LTP值从163.7±8.6% (n=7)减小到123.0±7.7% (n=4, P < 0.01),HNG预处理组与正常对照组(164.3±5.3%, n=7)非常接近(P>0.05),Genistein预处理组与Aβ25-35单独给予组(111.4±8.9%, n=7)非常相似。
以上结果显示,Aβ31-35和Aβ25-35具有相似的压抑海马LTP效应,提示Aβ31-35可能是Aβ更短的活性中心;HNG能够剂量依赖性地保护海马LTP免受Aβ引起的损伤,并且酪氨酸激酶通路可能参与了HNG的保护作用,提示应用HNG以及增强酪氨酸激酶活性可能成为治疗AD的有效方法或途径。
第二部分:[Gly14]-Humanin逆转了淀粉样β-蛋白对原代培养大鼠皮层神经元NMDA和GABAA受体电流的效应
兴奋性和抑制性突触活动的适度调节是大脑发挥正常生理功能的前提,兴奋性和抑制性受体/通道在突触传递过程特别是突触可塑性中起着关键作用。本实验在第一部分LTP实验发现的基础上,利用全细胞膜片钳技术,记录了原代培养的大鼠皮层神经元NMDA和GABAA受体介导的全细胞电流。通过重力灌流系统急性给予不同的Aβ片段、HNG及特异性酪氨酸激酶抑制剂Genistein,观察了不同的Aβ片段对神经元兴奋性和抑制性受体电流的效应,研究了神经保护肽HNG是否能对此效应产生调制作用及其可能的酪氨酸激酶机制。
实验结果显示:(1)单独应用高浓度(10μM)的Aβ25-35、Aβ31-35或Aβ35-31,均不能诱导出任何明显的跨膜电流。(2)急性给予0.1μM, 1μM和10μM Aβ25-35和Aβ31-35,可逆性地抑制了NMDA诱导的全细胞内向电流(INMDA),并表现出浓度依赖性,使Aβ25-35预处理组INMDA分别减小到86.0±6.7% (n=14, P<0.01), 74.9±8.2% (n=15, P<0.01)和61.0±10.6% (n=13, P<0.01);使Aβ31-35预处理组INMDA分别减小到82.6±8.0% (n=11, P<0.01), 72.1±10.8% (n=14, P<0.01)和60.6±7.8% (n=14, P<0.01),与给药前的(100%)相比均有统计学差异。但是,相同浓度的Aβ25-35和Aβ31-35抑制INMDA的效应没有显著性差异(P>0.05)。(3) HNG剂量依赖地逆转了10μM Aβ25-35和Aβ31-35对INMDA的抑制效应,INMDA从Aβ25-35单独给予时的61.0±10.6% (n=13)改变为共同给予0.1 nM, 1 nM和10 nM HNG时的58.1±5.5% (n=15, P.>0.05),80.7±8.8% (n=12, P<0.01)和96.5±6.0% (n=12, P<0.01);从Aβ31-35单独给予时的60.6±7.8% (n=14)改变为共同给予0.1 nM, 1 nM和10 nM HNG时的56.5±6.2% (n=15, P>0.05), 74.3±4.5% (n=14, P<0.01)和93.9±11.0% (n=13, P<0.01)。(4) 0.1μM, 1μM和10μMAβ25-35和Aβ31-35预处理,可逆性地抑制了GABA诱导的全细胞电流(IGABA),并表现出浓度依赖性。Aβ25-35预处理后IGABA分别减小到96.2±7.3% (n=12, P>0.05), 67.7±6.8% (n=14, P<0.01)和55.5±12.2% (n=13, P<0.01);Aβ31-35预处理后IGABA分别减小到91.0±8.0% (n=11, P<0.05), 71.3±9.1% (n=21, P<0.01)和58.2±9.7% (n=19, P<0.01),与给药前的对照(100%)相比都有统计学差异。同时,Aβ25-35和Aβ31-35抑制IGABA的效应,在相同浓度组之间也未发现显著性差异。(5) HNG剂量依赖性逆转了1μM Aβ25-35和Aβ31-35诱导的IGABA抑制效应。IGABA从Aβ25-35单独给予时的67.7±6.8%增加到共同给予0.01 nM,0.1 nM,1 nM和10 nM HNG时的67.8±9.6% (n=14, P>0.05), 80.8±10.8% (n=12, P<0.01), 90.2±10.9 (n=12, P<0.01)和99.6±9.4% (n=16, P<0.01);从Aβ31-35单独给予时的71.3±9.1%增加到共同给予0.01 nM,0.1 nM,1 nM和10 nM HNG时的66.9±8.8% (n=14, P>0.05), 83.8±8.4% (n=11, P<0.01), 92.2±8.8% (n=12, P<0.01)和102.5±9.7% (n=15, P<0.01)。(6) Aβ25-35和Aβ31-35对较高浓度(100μM,1000μM)GABA引起的全细胞电流没有显著影响。10μM,20μM或40μM Aβ25-35预处理后,100μM GABA引起的IGABA分别维持在99.9±5.9% (n=12, P>0.05),96.1±7.2% (n=11, P>0.05)和98.3±5.6% (n=12, P>0.05);类似地,10μM,20μM或40μM Aβ31-35预处理后,IGABA分别为97.8±5.3% (n=18, P>0.05), 99.3±9.5% (n=12, P>0.05)和95.1±9.6% (n=13, P>0.05)。与给药前(100%)相比均无显著性统计学差异。这表明,高浓度(100μM以上)GABA诱导的IGABA不易受到Aβ的抑制作用。(7)特异性酪氨酸激酶抑制剂Genistein (100μM)预处理后,几乎完全去除了HNG (10 nM)对Aβ25-35和Aβ31-35抑制INMDA和IGABA的保护作用。INMDA分别减小为63.0±8.0% (n=14)和58.9±4.8% (n=14);IGABA分别减小为70.5±11.4% (n=10)和70.5±11.4% (n=10)。与共同给予HNG和Aβ25-35/Aβ31-35相比有统计学差异,但与单独给予Aβ25-35或Aβ31-35相比无显著性统计学差异。(8)给予Aβ31-35的反序列即Aβ35-31预处理后,NMDA或GABA诱发的电流未出现明显改变。以上结果表明:急性给予Aβ25-35和Aβ31-35均可对原代培养的大鼠皮层神经元NMDA和GABA受体产生调制作用。这些结果可能有助于解释Aβ诱导的海马LTP的损害,其中突触内NMDA受体的下调可能会直接影响LTP的诱导;GABA受体的下调有可能破坏神经系统兴奋和抑制的平衡,增加细胞的兴奋毒作用。该结果为Aβ损伤谷氨酸能和GABA能系统以及HNG对抗Aβ神经毒性作用的保护效应提供了进一步的电生理证据。
第三部分:[Gly14]-Humanin对淀粉样β-蛋白引起的原代培养大鼠皮层神经元细胞内钙水平的影响
本实验使用激光扫描共聚焦显微镜成像系统进行细胞内Ca2+荧光成像实验,观察了各种Aβ片段对原代培养的大鼠皮层神经元细胞内Ca2+浓度(intracellular calciumconcentration, [Ca2+]i)的影响,并研究了HNG对Aβ诱导的钙稳态紊乱的保护作用及可能的酪氨酸激酶机制。
实验结果显示:(1) Aβ25-35或Aβ31-35均显著增加了原代培养的皮层神经元细胞内钙水平。细胞外给予25μM Aβ25-35或Aβ31-35处理18分钟后,神经元的相对荧光密度由给药前的对照值(100%)分别明显上升到174.3±10.7% (n=25, P<0.01)和172.7±6.6% (n=40, P<0.01);Aβ25-35和Aβ31-35升高[Ca2+]i的效应,在相同浓度时没有发现显著性差异;给予Aβ31-35的反序列Aβ35-31后,皮层神经元[Ca2+]i没有明显变化。(2) HNG阻断了Aβ25-35和Aβ31-35诱导的[Ca2+]i升高,并表现出一定程度的剂量依赖性。我们发现,用不同浓度的HNG预处理培养的神经元25～30 min后,随着HNG浓度的升高,Aβ25-35或Aβ31-35诱导的[Ca2+]i升高效应逐渐减小。在Aβ25-35组,0.1 nM, 1 nM, 10 nM和100 nM HNG预处理后,Aβ25-35 (25μM)引起的相对荧光密度由单独给予Aβ25-35时的174.3±10.7% (n=25)分别下降为173.6±9.0% (n=21, P>0.05)、144.6±8.2% (n=39, P<0.01)、120.0±6.3% (n=38, P<0.01)和105.4±6.0% (n=33, P<0.01);在Aβ31-35组,以上四种浓度的HNG预处理后,Aβ31-35 (25μM)引起的相对荧光密度则由单独给予Aβ31-35时的172.7±6.6% (n=40)分别下降为164.4±8.8% (n=16, P<0.01)、137.1±6.7% (n=19, P<0.01)、114.4±9.9% (n=23, P<0.01)和103.1±6.2% (n=29, P<0.01)。同单独使用Aβ25-35或Aβ31-35相比,联合使用HNG和Aβ25-35或Aβ31-35引起的[Ca2+]i下降具有明显差别。(3)特异性酪氨酸激酶抑制剂Genistein (100μM)预处理后,基本上去除了HNG (10 nM)对Aβ25-35或Aβ31-35 (25μM)引起的[Ca2+]i升高效应的抑制作用。与联合给予HNG和Aβ25-35/Aβ31-35相比,共同给予Genistein、HNG和Aβ25-35/Aβ31-35后,相对荧光密度值分别从120.0±6.3% (n=38)和114.4±9.9% (n=23)增加到178.3±9.9% (n=29, P<0.01)和168.7±8.2% (n=27, P<0.01),并且与单独给予Aβ25-35或Aβ31-35的相对荧光密度值相似,意味着HNG的保护作用被酪氨酸激酶抑制剂所阻断。
钙成像实验结果表明:(1) Aβ25-35和Aβ31-35均可使原代培养的大鼠皮层神经元[Ca2+]i升高,提示Aβ的神经毒性与Aβ引起的细胞内钙超载有关;(2) HNG剂量依赖地抑制了Aβ25-35和Aβ31-35诱导的[Ca2+]i升高,提示HNG对原代培养的大鼠皮层神经元具有保护作用,其机制至少部分是通过减弱Aβ引起的细胞内钙超载实现的;(3)特异性酪氨酸激酶抑制剂Genistein去除了HNG对Aβ25-35或Aβ31-35引起的[Ca2+]i升高的抑制作用,提示某些酪氨酸激酶参与了HNG对Aβ诱导的[Ca2+]i升高的保护作用;(4) Aβ25-35和Aβ31-35引起[Ca2+]i升高的效应在许多实验组中都非常相似,包括Aβ片段单独使用和Aβ与HNG、Genistein联合使用,这支持我们之前提出的假设:Aβ31-35是全长Aβ分子中一个更短的片段,可能是Aβ分子发挥神经毒性作用的活性中心。
总之,本研究利用电生理技术即细胞外场电位记录和全细胞膜片钳记录手段,结合激光扫描共聚焦显微镜Ca2+荧光成像技术,通过记录大鼠在体海马LTP、引导原代培养的皮层神经元NMDA受体和GABA受体电流以及测定神经元细胞内钙离子水平的变化,探讨了神经保护肽HNG对Aβ神经毒性作用的调制以及可能的酪氨酸激酶机制。研究结果首次表明,HNG能够保护神经毒性Aβ诱导的在体海马LTP损伤、逆转Aβ对LTP相关受体(NMDA和GABA受体)电流的损害、并且抑制了Aβ引起的[Ca2+]i升高。我们进一步的结果还提示,酪氨酸激酶通路可能参与了HNG的神经保护作用。因此,本研究为HN的神经保护作用及其机制提供了强有力的电生理和细胞内钙成像证据,为HNG有可能成为AD治疗措施之一提供了研究基础。
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elderly leading to progressive loss of memory and cognitive deficits. One of the predominant neuropathological features of AD is the presence of high density of senile plaques in the brain. The main constituent of senile plaques is amyloidβ-protein (Aβ), which consisting of 39-43 amino acids and coming from the proteolysis of amyloid precursor protein (APP). The neurotoxicity of Aβhas been widely reported in vivo and in vitro, including the impairment of synaptic plasticity, such us long-term potentiation (LTP). The mechanisms underlying Aβ-neurotoxicity are complex and unclear so far but may involve the vulnerability of glutamatergic and GABAergic system. Considerable evidences suggest that Aβand its peptide fragments influence cellular homeostasis and neuronal signaling through modulation of ion channel function.
Hippocampus in the central nervous system has been widely considered to be a crucial center for learning and memory. Hippocampal LTP is an activity-dependent increase in the synaptic response and has been accepted as a popular electrophysiological model for the cellular basis of learning and memory, owing to the close relationship between the hippocampal LTP impairment and the animal cognitive deficient. Further, N-methyl-D-aspartate (NMDA) receptor plays a pivotal role in the LTP induction of CA1 pyramidal neurons because of its high permeability to Ca2+ ions; changes in the efficacy of synaptic inhibition mediated by GABAA receptors are believed to play central roles in certain forms synaptic plasticity, and some reports indicate the potential role of Aβin the deficits of the GABAergic system in the development and pathology progresses of AD. Hence the modulation of LTP-related receptors such as NMDA and GABA receptors is a potential target for the neurotoxicity of Aβon LTP. At the same time, calcium ion is one of the most important intracellular second messengers in the brain, being essential for a variety of neuronal functions such as neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways. It is well known that Aβinduced Ca2+ influx is proposed as the initial event of Aβ-induced multiple events. Although the mechanisms by which Aβincreased [Ca2+]i are still not well known, the disruption of Ca2+ homeostasis is the primary event in Aβneurotoxicity. Thus, it will be very important to search for effective medicines or measures that can protect NMDA receptors, GABA receptors, and intracellular Ca2+ homeostasis against Aβ-induced neurotoxicity in the prevention and treatment of AD.
An important clue in the development of AD therapy is the finding of humanin (HN), a novel short polypeptide, and its potent derivative [Gly14]-humanin (HNG). HN can prevent neuronal cell death caused by various AD-relevant insults such as neurotoxic Aβprotein, and is the only factor that is effective in suppressing various types of AD-related neuronal death so far compared with many neurotrophic factors. The fact that HNG reverses the impairment of memory induced by Aβpeptide suggests that HNG is a promising candidate in the treatment of AD. However, it is still short of in vivo and in vitro electrophysiological evidence for the protection of HN on synaptic plasticity such as LTP and LTP-related postsynaptic receptors/channels, and the molecular mechanisms that underling the neuroprotective function of HN also remain largely unknown.
Therefore, the purposes of the present study were as follows: (1) Examining the effects of intracerebroventricular (i.c.v.) injection of HNG on the different Aβfragments-induced suppression of LTP in the rat hippocampal CA1 region in vivo with field potential recording technique; (2) Investigating whether HNG affects the effects of different Aβfragments on LTP induction-related postsynaptic ligand-gated excitatory receptor channels (NMDAR) and inhibitory receptor channels (GABAR) by using whole-cell patch clamp technique in cultured primary rat cortical neurons; (3) Observing the effects of different Aβfragments on [Ca2+]i, and investigating the possible neuroprotective effects of HNG against Aβ-induced disruption of Ca2+ homeostasis in cultured primary rat cortical neurons by using calcium imaging via laser-scanning confocal imaging system; (4) Clarifying whether tyrosine kinases are involved in the neuroprotective function of HNG by using a specific tyrosine kinase inhibitor, Genistein; and (5) Identifying whether Aβ31-35 is a shorter active center than Aβ25-35 in full length of Aβmolecule by comparing the effects of Aβfragments in the experiments of LTP, whole-cell currents and [Ca2+]i imaging.
PartⅠ:[Gly14]-Humanin Rescues Long-Term Potentiation from AmyloidβProtein-Induced Impairment in the Rat Hippocampal CA1 Region In Vivo
The present study observed the effects of intracerebroventricular (i.c.v.) injection of HNG on the different Aβfragments-induced suppression of hippocampal LTP in area CA1 of urethane anesthetized rats, and explored the probable tyrosine kinase mechanism by which HNG protect Aβ-induced LTP impairment. The results showed that: (1) The amplitudes of the field excitatory postsynaptic potentials (fEPSPs) in control group (i.c.v. injection of saline) increased to 215.1±8.6% immediately after applying high-frequency stimulus (HFS), as compared to that measured before HFS and set arbitrarily as 100%, and remains at 163.3±8.5% 60 min after HFS (n=8). (2) i.c.v. injection of 20 nmol Aβ25-35 and Aβ31-35 had no effect on baseline synaptic transmission, but both of them similarly and significantly depressed the induction of LTP, with fEPSPs of 111.4±8.9% (n=7) and 103.3±7.4% (n=6) at 60 min after HFS, respectively. (3) Application of 5 nmol HNG alone caused no significant effect on baseline synaptic transmission and HFS-induced LTP compared with control, and the LTP at 60 min post HFS was 159.2±6.4%(n=5), which is close to the value of 163.3±8.5% (n=8) found in control group (P>0.05). However, co-injection of HNG and Aβ25-35 reversed the depression of LTP by Aβ25-35 alone in a dose-dependent manner. After i.c.v. co-injection of 0.2 nmol, 1 nmol and 5 nmol HNG with 20 nmol Aβ25-35, the LTP at 60 min post HFS increased from 111.4±8.9% (n=7) in Aβ25-35 alone group to 114.9±6.4% (n=9, P>0.05), 130.9±6.5% (n=8, P<0.01) and 163.7±8.6% (n=7, P<0.01) in three HNG groups, respectively. (4) Similar to Aβ25-35, co-injection of 5 nmol HNG with 20 nmol Aβ31-35 did not affect baseline synaptic transmission, but effectively reversed Aβ31-35-induced LTP suppression, and the LTP value was 158.6±6.1% (n=5) 60 min after HFS, significantly larger than the effect when using 20 nmol Aβ31-35 alone (103.3±7.4%, n=6, P<0.01). (5) Separate application or co-application of HNG and Aβ25-35 showed no significant effect on paired pulse-evoked facilitation. (6) Pretreatment with 200 nmol Genistein obviously attenuated the protection of HNG against Aβ25-35-induced LTP impairment, and the LTP value decreased from 163.7±8.6% (n=7) to 123.0±7.7% (n=4, P<0.01) 60 min after HFS. The former was very close to the value in normal control group (164.3±5.3%, n=7, P>0.05), while the later has been close to the value in Aβ25-35 alone group (111.4±8.9%, n=7, P>0.05).
These results demonstrate that: (1) Aβ31-35 may be a shorter active fragment of Aβbecause of the similar effects of Aβ31-35 and Aβ25-35; (2) HNG could dose-dependently protect against the neurotoxic Aβ-induced hippocampal LTP impairment; and (3) the tyrosine kinase pathway was involved in the neuroprotective action of HNG. All of these results suggest that HNG might be one of the promising candidates for the treatment of AD in the future.
Part II:[Gly14]-Humanin Abolishes the Effects of AmyloidβProtein on NMDA and GABAA Receptor-Mediated Currents in Cultured Primary Rat Cortical Neurons
The proper modulation of excitatory and inhibitory synaptic transmission is critical for brain function including synaptic plasticity such as LTP. The present study extended our investigation of in vivo LTP by observing whether HNG can modulate the effects of different Aβ fragments on LTP induction-related postsynaptic ligand-gated excitatory receptor channels (NMDAR) and inhibitory receptor channels (GABAR) by using whole-cell patch clamp in cultured primary rat cortical neurons. The results showed that: (1) Acute application of Aβ25-35, Aβ31-35 or Aβ35-31 alone did not induce any membrane current, even at a higher concentration (10μM). (2) The NMDA-induced whole cell currents (INMDA) were reversibly inhibited by acute application of 0.1μM, 1μM and 10μM Aβ25-35 and Aβ31-35 in a dose-dependent manner. INMDA decreased to 86.0±6.7% (n=14, P<0.01), 74.9±8.2% (n=15, P<0.01) and 61.0±10.6% (n=13, P<0.01) in Aβ25-35 groups, 82.6±8.0% (n=11, P<0.01), 72.1±10.8% (n=14, P<0.01) and 60.6±7.8% (n=14, P<0.01) in Aβ31-35 groups compared with 100% in control, respectively. (3) HNG dose-dependently reversed the inhibitory effect of Aβ25-35 and Aβ31-35 (10μM) on INMDA. INMDA changed from 61.0±10.6% (n=13) in Aβ25-35 alone group to 58.1±5.5% (n=15, P>0.05), 80.7±8.8% (n=12, P<0.01) and 96.5±6.0% (n=12, P<0.01) at 0.1 nM, 1 nM and 10 nM HNG, respectively; and from 60.6±7.8% (n=14) in Aβ31-35 alone group to 56.5±6.2% (n=15, P>0.05), 74.3±4.5% (n=14, P<0.01) and 93.9±11.0% (n=13, P<0.01), respectively. (4) Meanwhile, pretreatment with 0.1μM, 1μM and 10μM Aβ25-35 and Aβ31-35 reversibly suppressed the GABA-induced whole-cell currents (IGABA) in a concentration-dependent manner. IGABA decreased to 96.2±7.3% (n=12, P>0.05), 67.7±6.8% (n=14, P<0.01) and 55.5±12.2% (n=13, P<0.01) in Aβ25-35 groups, 91.0±8.0% (n=11, P<0.05), 71.3±9.1% (n=21, P<0.01) and 58.2±9.7% (n=19, P<0.01) in Aβ31-35 groups compared with 100% in control, respectively. (5) HNG concentration-dependently reversed Aβ-induced suppression. IGABA changed from 67.7±6.8% in Aβ25-35 alone group to 67.8±9.6% (n=14, P>0.05), 80.8±10.8% (n=12, P<0.01), 90.2±10.9 (n=12, P<0.01) and 99.6±9.4% (n=16, P<0.01) at 0.01 nM, 0.1 nM,? 1 nM and 10 nM HNG, respectively. IGABA changed from 71.3±9.1% in Aβ31-35 alone group to 66.9±8.8% (n=14, P>0.05), 83.8±8.4% (n=11, P<0.01), 92.2±8.8% (n=12, P<0.01) and 102.5±9.7% (n=15, P<0.01) at 0.01 nM, 0.1 nM,? 1 nM and 10 nM HNG pretreatment, respectively. (6) Aβ25-35 and Aβ31-35 have no significant effect on the higher concentration (100μM and 1000μM) of GABA-induced whole cell currents. 100μM GABA induced IGABA were still 99.9±5.9% (n=12, P>0.05), 96.1±7.2% (n=11, P>0.05) and 98.3±5.6% (n=12, P>0.05) after application of 10μM, 20μM or 40μM Aβ25-35. Similarly, after pretreatment with 10μM, 20μM or 40μM Aβ31-35, 100μM GABA induced IGABA were still 97.8±5.3% (n=18, P>0.05), 99.3±9.5% (n=12, P>0.05) and 95.1±9.6% (n=13, P>0.05), respecitively. (7) Pretreatment with Genistein (100μM), a tyrosine kinase inhibitor, nearly completely abolished the protective action of HNG (10 nM) on Aβ25-35 and Aβ31-35 induced inhibition of INMDA and IGABA, the values being 63.0±8.0% (n=14, P<0.01) and 58.9±4.8% (n=14) for INMDA, 70.5±11.4% (n=10) and 70.5±11.4% (n=10) for IGABA. (8) Aβ35-31, the reversed sequence of Aβ31-35, showed no effect on the currents induced by NMDA or GABA.
In conclusion, the present study showed an acute modulation of Aβ25-35 and Aβ31-35 on NMDA receptors and GABAA receptors in cultured primary rat cortical neurons. These results may partly explain the reason of Aβ-induced suppression of hippocampal LTP, in which synaptic NMDA receptor might play important roles. In addition, our results provide an electrophysiological evidence for the Aβ-neurotoxicity on the glutamatergic/GABAergic system and the neuroprotective function of HNG on Aβ-neurotoxicity. The neuroprotection of HNG strongly suggests the potential clinical applications of HNG in novel AD therapies.
PartⅢ:Effects of [Gly14]-Humanin on AmyloidβProtein-Induced Calcium Influx in Cultured Primary Rat Cortical Neurons
The present study, by utilizing calcium imaging via laser-scanning confocal imaging system, observed the effects of different Aβfragments on [Ca2+]i, and investigated the neuroprotective effects of HNG against Aβ-induced disruption of Ca2+ homeostasis and its possible tyrosine kinase mechanism in cultured primary rat cortical neurons. The results showed that: (1) Both Aβ25-35 and Aβ31-35 significantly increased the intracellular calcium levels of the cultured primary rat cortical neurons. After application of 25μM Aβ25-35 or Aβ31-35 to cultured primary rat cortical neurons, the relative fluorescent intensity of neurons obviously increased from 100% in control to 174.3±10.7% (n=25, P<0.01) and 172.7±6.6% (n=40, P<0.01), respectively. However, 25μM Aβ35-31, the reverse peptide of Aβ31-35, had no effect on [Ca2+]i elevation. In addition, the effect of Aβ25-35 and Aβ31-35 on [Ca2+]i elevation, at the same concentration, does not show any statistical difference. (2) Neuroprotective polypeptide HNG blocked the [Ca2+]i elevation induced by Aβ25-35 or Aβ31-35 in a concentration-dependent manner. With the increasing of HNG concentration, Aβ25-35- or Aβ31-35-induced elevation in [Ca2+]i decreased. After pretreatment with 0.1 nM, 1 nM, 10 nM and 100 nM HNG, Aβ25-35-evoked relative fluorescent intensity decreased from 174.3±10.7% (n=25) to 173.6±9.0% (n=21, P>0.05), 144.6±8.2% (n=39, P<0.01), 120.0±6.3% (n=38, P<0.01), and 105.4±6.0% (n=33, P<0.01), respectively. Aβ31-35-evoked relative fluorescent intensity decreased from 172.7±6.6% (n=40) in Aβ31-35 alone to 164.4±8.8% (n=16, P<0.01), 137.1±6.7% (n=19, P<0.01), 114.4±9.9% (n=23, P<0.01) and 103.1±6.2% (n=29, P<0.01), respectively. (3) Pretreatment with Genistein (100μM), a specific tyrosine kinase inhibitor, essentially abolished the protective effect of HNG (10 nM) on Aβ25-35 or Aβ31-35 induced [Ca2+]i elevation. Compared with co-application of HNG and Aβ25-35 or Aβ31-35, the relative fluorescent intensity in co-application of Genistein, HNG and Aβ25-35 or Aβ31-35 increased to 178.3±9.9% (n=29, P<0.01) and 168.7±8.2% (n=27, P<0.01) from 120.0±6.3% (n=38) and 114.4±9.9% (n=23), respectively, similar to the values in Aβ25-35 or Aβ31-35 alone group.
These results demonstrate that: (1) Both Aβ25-35 and Aβ31-35 can similarly elevate [Ca2+]i in cultured primary rat cortical neurons, suggesting that Aβ-neurotoxicity is related with Aβ-induced intracellular calcium overloading. (2) HNG can inhibit Aβ25-35 and Aβ31-35-induced elevation of [Ca2+]i in a concentration-dependent manner, suggesting that the protective action of HNG on the cultured primary rat cortical neurons was, at least in part, mediated by attenuating the intracellular calcium overloading induced by Aβ. (3) Genistein, a specific tyrosine kinase inhibitor, abolished the suppression of HNG on Aβ25-35 or Aβ31-35 induced [Ca2+]i elevation, indicating that certain tyrosine kinase(s) is (are) involved in the protective effects of HNG on Aβinduced [Ca2+]i elevation. (4) The similar [Ca2+]i elevation effects of Aβ31-35 and Aβ25-35 in various groups, including Aβfragments alone and Aβplus HNG and Genistein, supports the hypothesis we proposed previously that Aβ31-35 may be a more shorter fragment of the full length of Aβmolecule.
In conclusion, the present study, by using field potential recording and whole-cell patch clamp technique, observed the modulation of HNG on the effects of Aβfragments on hippocampal LTP in vivo, INMDA and IGABA in cultured primary rat cortical neurons. The possible mechanisms underlying the neuroprotection of HNG were also explored by using Genistein and Ca2+ image technique. The results demonstrated for the first time that HNG could protect against the neurotoxic Aβ-induced hippocampal LTP deficit, reverse the impairment of LTP-related NMDA and GABA receptor currents by Aβand inhibit Aβ-induced [Ca2+]i elevation. Moreover, our results indicated that the neuroprotective actions of HNG may be involved in the tyrosine kinase pathway. Taken all together, all of our experiments in vivo and in vitro provide strong electrophysiological evidences for the neuroprotection of HN and suggest that HNG might be one of the promising candidates for the treatment of AD in the future.
引文
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